16S V3-V4 amplicon sequencing of Medicago truncatula root and root nodule tissues
We extracted and purified genomic DNA from tissue samples using a DNeasy Plant Pro Kit (Qiagen, Hilden, Germany) following the manufacturers instructions. For each sample, we evaluated the DNA concentration using optical density measurements obtained by a Nanodrop spectrophotometer (NanoDrop Technologies, Rockland, DE, USA) at 260 and 280 nm wavelengths. We submitted DNA samples to the W.M. Keck Center for Comparative and Functional Genomics at the University of Illinois at Urbana-Champaign for 16S rRNA V3-V4 region amplicon sequencing. Briefly, DNA quality was assessed with a Qubit fluorometer (Invitrogen, MA, USA) and the targeted 16S regions were amplified by PCR using primers V3-F357_N (5-CCTACGGGNGGCWGCAG-3) and V4-R805 (5-GACTACHVGGGTATCTAATCC-3) using a Fluidigm Biomark HD PCR machine (Fluidigm Corporation, South San Francisco, CA, USA). PCR products were quantified with a QuantiT PicoGreen fluorometer (Invitrogen, MA, USA), then cleaned, purified, and size-selected in a 2% agarose E-gel (Invitrogen, MA, USA) followed by gel extraction with a QIAquick Gel extraction kit (Qiagen, Hilden, Germany). Amplicons were sequenced with Illumina MiSeq v2 platform (Illumina, San Diego, CA, USA) according to the manufacturers instructions.